野生型和突变型荞麦蛋白酶抑制剂的活性比较及抗肿瘤功能分析

运用定点突变技术研究重组荞麦胰蛋白酶抑制剂(rBTI)的作用位点,先后构建了R45A-aBTI 和R45F-fBTI 两个突变体．抑制活性测定显示,aBTI 和fBTI 均丧失了胰蛋白酶抑制活性,却分别增加了对弹性蛋白酶和胰凝乳蛋白酶的抑制活性,确定Arg⁴⁵为野生型rBTI 的作用位点．稳定性分析表明,rBTI、aBTI 和fBTI 均具有很高的热稳定性及酸碱稳定性．采用MTT 比色法分别检测野生型和突变型抑制剂对肿瘤细胞生长的抑制作用．结果表明,突变前后的3种抑制剂对HL-60 和EC9706 细胞的生长均显示出很强的抑制作用,并且具有明显的浓度依赖性和时间依赖性．通过研究不仅确定了rBTI 的作用位点,而且获得了两种新型蛋白酶抑制剂,且作用位点的改变并不影响其对肿瘤细胞的生长抑制作用．为进一步研究胰蛋白酶抑制剂的结构与功能的关系以及抗肿瘤药物的研制提供了新的思路．     

复方木鸡冲剂诱导人白血病细胞HL-60凋亡机制研究

目的:探讨复方木鸡冲剂诱导人白血病细胞HL-60细胞凋亡的作用和机制,为相关中药开发提供实验资料.方法:用MTT法检测复方木鸡冲剂对HL-60细胞增殖活性的影响,光镜下观察细胞形态的变化;流式细胞仪(Annexin V/PI双染法)检测细胞凋亡,并分析细胞周期;免疫细胞化学法检测Bcl-2、caspase-3、p21WAF1的表达.结果:MTT法显示复方木鸡冲剂能抑制HL-60细胞的生长,细胞呈凋亡形态学变化.流式细胞仪检测结果为细胞凋亡率明显增高,出现GO/G1期阻滞.Bcl-2表达降低,caspase-3表达增高,p21WAF1表达强阳性.结论:复方木鸡冲剂明显抑制HL-60细胞的生长,其抗肿瘤的机制与诱导肿瘤细胞凋亡、促进细胞分化有关.     

9R穿膜肽可增强P53抗肿瘤效果

为解决P53蛋白难以进入细胞内部发挥治疗作用的瓶颈难题.将p53基因融合插入带有9个精氨酸作为穿膜肽的表达载体中表达融合蛋白CPPs-P53,并与没有穿膜肽的P53蛋白进行比较,利用Western blotting方法检测蛋白的表达情况,MTT及Annexin V/PI双染法检测细胞生长抑制率及细胞凋亡率.Western blotting检测表明已成功在原核表达系统中表达融合蛋白CPPs-P53和P53蛋白,且蛋白纯度均已达到90％以上;MTT检测表明,P53蛋白对肿瘤细胞的生长虽有一定的抑制作用,但融合蛋白CPPs-P53与之相比,对肿瘤细胞生长的抑制效果显著增强,细胞生长抑制率有明显的提升,并且细胞生长抑制率呈现剂量依赖性;Annexin V/PI双染检测细胞凋亡情况也表明P53虽可以在一定程度上诱导肿瘤细胞的凋亡,但与P53蛋白相比较,融合蛋白CPPs-P53诱导的凋亡细胞明显增加,凋亡率是P53蛋白的2～3倍.由此说明在抑制肿瘤细胞的生长和诱导细胞凋亡方面,CPPs-P53比没有穿膜肽的P53蛋白的效果更显著.     

重组荞麦蛋白酶抑制剂aBTI的表达及其对肝癌HepG2细胞增殖的影响

为了深入研究不同类型荞麦蛋白酶抑制剂在抑制肿瘤细胞生长及诱导其发生凋亡方面的作用,本实验在先前得到的重组荞麦胰蛋白酶抑制剂(rBTI)的基础上,运用定点突变技术将rBTI的活性位点进行替换,构建一种新型蛋白酶抑制剂aBTI。通过在大肠杆菌M15[pREP4]中表达,获得以可溶形式存在的aBTI目的蛋白。经Ni~(2+)-NTA亲和层析及superdex 75分子筛层析纯化,目的蛋白的纯度达到95%以上。抑制活性测定表明,aBTI具有专一性的弹性蛋白酶抑制活性,抑制常数K_i为3.34×10~(-7)mol/L。MTT比色法检测及细胞核形态学观察显示,aBTI在体外能够显著抑制HepG2肿瘤细胞的增殖(IC_(50):1.88μmol/L),并诱导其凋亡,具有较好的抗肿瘤效应。     

灰毡毛忍冬次皂苷乙抑制白血病细胞HL-60的增殖及其机制研究

研究了灰毡毛忍冬次皂苷乙(MB)在体外对白血病细胞HL-60和结肠癌细胞LOVO增殖的抑制作用,并初步探讨其分子机制。采用MTT法检测MB的增殖抑制作用;利用RT2ProfilerTMPCR Array芯片实时定量PCR扩增肿瘤发生中84个关键基因。结果表明MB对两种肿瘤细胞生长均有抑制作用,且对HL-60效果更好。以HL-60作为细胞模型,总共发现差异基因20个,其中上调基因14个,下调基因6个,主要作用是阻滞细胞周期和降低细胞侵袭转移。     

氟苯达唑对人急性髓系白血病HL-60细胞的增殖抑制作用研究

目的:研究氟苯达唑对人急性髓系白血病HL-60细胞增殖的抑制作用,明确氟苯达唑对HL-60细胞周期,凋亡发生的作用机制。方法:噻唑蓝法(MTT)检测氟苯达唑对人急性髓系白血病HL-60细胞的生长抑制作用,流式细胞术检测氟苯达唑对HL-60细胞周期,DNA片段化的影响,免疫印迹法检测Caspase, Raf, Bcl-2家族蛋白表达。结果:氟苯达唑抑制人急性髓系白血病HL-60细胞生长,HL-60细胞G2/M期增加,与阴性对照组相比,在一定的剂量和时间内,差别具有显著统计学意义;DNA片段化上升,0.25,0.5,1μM组与对照组相比差别具有显著统计学意义,促使Cleaved PARP,Cleaved-caspase 3,Cleaved-caspase 9蛋白表达量趋势增加;Bag-1和Bcl-2蛋白表达量降低;b-raf,c-raf磷酸化蛋白表达水平逐渐降低。结论:氟苯达唑通过诱导HL-60细胞阻滞于G2/M期,增加DNA片段化水平,激活Caspase, Raf, Bcl-2家族介导的凋亡相关通路抑制人急性髓系白血病HL-60细胞增殖,诱导人急性髓系白血病HL-60细胞发生凋亡而发挥抗肿瘤作用。     

沉默FNBP1基因对人HL-7702细胞体外增殖及迁移能力的影响

为了研究沉默FNBP1(formin-binding protem1)基因对细胞体外增殖以及对细胞迁移能力的影响,设计并构建了3个靶向FNBP1 siRNA干扰载体(Si-1,Si-2,Si-3),经酶切和DNA测序鉴定后转染人正常的肝细胞HL-7702。RT-PCR和Western blot检测瞬时转染细胞中FNBP1基因的干扰效率,筛选出特异高效的RNAi靶点。并用G418筛选稳定表达了RNAi的细胞株,用XTT法检测细胞体外增殖率,划痕法检测细胞的迁移能力。结果显示:酶切和测序结果证明干扰质粒构建正确;转染特异性的干扰质粒(Si-1、Si-2、Si-3)细胞的FNBP1基因表达量与对照组相比均有所降低,在蛋白质水平表达也明显受到抑制,且重组干扰质粒Si-2的干扰效应较强。XTT检测结果表明,构建的沉默FNBP1重组质粒Si-2能显著抑制HL-7702细胞的体外增殖能力,但并不直接影响HL-7702细胞的迁移能力。     

一株新发现新城疫病毒体外高效杀伤肝癌细胞作用机制初探

目的:与NDV疫苗株LaSota对比研究一株NDV D817株对肝癌细胞高效特异性的杀伤效应和作用机制,进一步筛选NDV溶瘤毒株.方法:用MTT法对比病毒对三株传代肝癌细胞株SMMC-7721、Bel-7404和HepG-2及一株正常肝细胞株HL-7702的杀伤效应,并用TUNNL法及透射电镜观察病毒诱导肿瘤细胞发生凋亡作用.结果:NDV D817株对肝癌细胞株SMMC-7721、Bel-7404和HepG-2杀伤效应高达80%,显著高于疫苗LaSota株(P<0.01),而对人正常肝细胞HL-7702无明显影响;病毒在肝癌细胞中明显复制增殖,对细胞的杀伤活性与病毒作用剂量和病毒作用时间成正比;NDV D817株有效诱导肝癌细胞发生凋亡.结论:NDV D817株有效诱导肝癌细胞发生凋亡,对SMMC-7721、Bel-7404和HepG-2细胞具有高效杀伤性,而对正常肝细胞HL-7702未见明显影响.推测为溶瘤株.     

塞来昔布联合氟尿嘧啶对胰腺癌细胞株SW1990生长抑制作用及其机制研究

目的:研究选择性环氧化酶-2(COX-2)抑制剂塞来昔布联合氟尿嘧啶对胰腺癌细胞株SW1990生长的抑制作用,并对其机制进行初步探讨.方法:实验分为对照组、氟尿嘧啶组、塞来昔布组及两药联合组,将不同浓度的药物分剐作用于胰腺癌细胞株SW1990,MTT法检测各组细胞的生长抑制率,并探索产生最佳细胞生长抑制作用的药物浓度.流式细胞仪检测不同药物作用对肿瘤细胞周期的影响.RT-PCR检测各组细胞Survivin的表达情况.结果:MTT法显示氟尿嘧啶、塞来昔布均能抑制SW1990的生长,且细胞的存活率都随药物浓度的增加而降低.两药联合组对细胞生长的抑制作用更明显.流式细胞仪检测结果显示塞来昔布组、氟尿嘧啶组及两药联合组细胞较对照组G0/G1期细胞比例明显增加,S期和G2/M期细胞比例明显减少.RT-PCR结果显示氟尿嘧啶组、塞来昔布组、联合组都可下调Survivin的表达,以联合组最为明显.结论:塞来昔布联合氟尿嘧啶对胰腺癌SW1990细胞的生长具有抑制作用,其机制可能与下调Survivin的表达从而诱导细胞的凋亡和细胞周期的停滞有关.     

新生牛肝中三种低分子化合物牛磺酸、鸟氨酸、肌肽对HL-60细胞凋亡的诱导作用

目的:研究三种新生牛肝源低分子化合物:牛磺酸、鸟氨酸、肌肽对HL-60白血病细胞增殖的抑制作用并探讨其调控机理.方法:用MTT法分别检测三种活性成分作用后HL-60细胞和正常人淋巴细胞的存活率.分别用琼脂糖凝胶电泳,顺磁共振ESR技术,免疫组化法测定其对HL-60细胞的核DNA、氧自由基活性和细胞周期蛋白水平的影响.结果:三种化合物能够有效地抑制HL-60细胞的增殖,而对正常的人淋巴细胞的生长没有抑制作用;三种化合物使HL-60细胞的核DNA产生30 kb片段,使HL-60细胞内的氧自由基活性降至痕量水平,并下调HL-60的p45/skp2的水平,而上调p27/kip的水平.结论:牛磺酸、鸟氨酸、肌肽能够通过调控细胞周期蛋白的水平而选择性的抑制肿瘤细胞的增殖.     

Effects of rapamycin on cell proliferation and phosphorylation of mTOR and p70(S6K) in HepG2 and HepG2 cells overexpressing constitutively active Akt/PKB

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that plays an important role in the regulation of cell proliferation and protein synthesis through the activation of its downstream target ribosomal p70 S6 kinase (p70(S6K)). The levels of p-mTOR are regulated by the protein kinase B (Akt/PKB). Therefore, the effects of insulin and rapamycin (an inhibitor of mTOR) on the phosphorylation of mTOR (Ser 2448) and p70(S6K) (Thr 389) as well as on cell proliferation in parental HepG2 cells and HepG2 cells overexpressing constitutively active Akt/PKB (HepG2-CA-Akt/PKB) were studied. Insulin increased the levels of phosphorylated mTOR and p70(S6K) in both the cell lines. Rapamycin treatment partially decreased the phosphorylation of mTOR but completely abolished the phosphorylation of p70(S6K) in the absence as well as presence of insulin in both cell lines. The effect of insulin and rapamycin on the cell proliferation in both cell lines was further studied. In the presence of serum, parental HepG2 cells and HepG2-CA-Akt/PKB showed an increase in cell proliferation until 120 and 168 h respectively. Rapamycin inhibited cell proliferation under all experimental conditions more evident under serum deprived conditions. Parental HepG2 cells showed decline in the cell proliferation after 48 h and the presence of insulin prolonged cell survival until 120 h and this effect were also inhibited by rapamycin under serum deprived conditions. On the contrary, HepG2-CA-Akt/PKB cells continued proliferation until 192 h. The effects of insulin on cell proliferation were more pronounced in parental HepG2 cells as compared to HepG2-CA-Akt/PKB cells. Long term effects of rapamcyin significantly decreased the levels of p-mTOR (Ser 2448) both in the presence and absence of insulin in these cells. A positive correlation between the levels of p-mTOR (Ser2448) and cell proliferation was observed (99% confidence interval, r(2)=0.525, p<0.0001). These results suggest that rapamycin causes a decline in the cell growth through the inhibition of mTOR.     

ETOH inhibits embryonic neural stem/precursor cell proliferation via PLD signaling

While a mother’s excessive alcohol consumption during pregnancy is known to have adverse effects on fetal neural development, little is known about the underlying mechanism of these effects. In order to investigate these mechanisms, we investigated the toxic effect of ethanol (ETOH) on neural stem/precursor cell (NSC) proliferation. In cultures of NSCs, phospholipase D (PLD) is activated following stimulation with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Exposure of NSCs to ETOH suppresses cell proliferation, while it has no effect on cell death. Phosphatidic acid (PA), which is a signaling messenger produced by PLD, reverses ETOH inhibition of NSC proliferation. Blocking the PLD signal by 1-butanol suppresses the proliferation. ETOH-induced suppression of NSC proliferation and the protective effect of PA for ETOH-induced suppression are mediated through extracellular signal-regulated kinase signaling. These results indicate that exposure to ETOH impairs NSC proliferation by altering the PLD signaling pathway.     

Inhibition of cellular proliferation through IkappaB kinase-independent and peroxisome proliferator-activated receptor gamma-dependent repression of cyclin D1

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-regulated nuclear receptor superfamily member. Liganded PPARgamma exerts diverse biological effects, promoting adipocyte differentiation, inhibiting tumor cellular proliferation, and regulating monocyte/macrophage and anti-inflammatory activities in vitro. In vivo studies with PPARgamma ligands showed enhancement of tumor growth, raising the possibility that reduced immune function and tumor surveillance may outweigh the direct inhibitory effects of PPARgamma ligands on cellular proliferation. Recent findings that PPARgamma ligands convey PPARgamma-independent activities through IkappaB kinase (IKK) raises important questions about the specific mechanisms through which PPARgamma ligands inhibit cellular proliferation. We investigated the mechanisms regulating the antiproliferative effect of PPARgamma. Herein PPARgamma, liganded by either natural (15d-PGJ(2) and PGD(2)) or synthetic ligands (BRL49653 and troglitazone), selectively inhibited expression of the cyclin D1 gene. The inhibition of S-phase entry and activity of the cyclin D1-dependent serine-threonine kinase (Cdk) by 15d-PGJ(2) was not observed in PPARgamma-deficient cells. Cyclin D1 overexpression reversed the S-phase inhibition by 15d-PGJ(2). Cyclin D1 repression was independent of IKK, as prostaglandins (PGs) which bound PPARgamma but lacked the IKK interactive cyclopentone ring carbonyl group repressed cyclin D1. Cyclin D1 repression by PPARgamma involved competition for limiting abundance of p300, directed through a c-Fos binding site of the cyclin D1 promoter. 15d-PGJ(2) enhanced recruitment of p300 to PPARgamma but reduced binding to c-Fos. The identification of distinct pathways through which eicosanoids regulate anti-inflammatory and antiproliferative effects may improve the utility of COX2 inhibitors.     

Differential effects of glucocorticoids on the proliferation of a murine helper and a cytolytic T cell clone in response to IL-2 and IL-4

The proliferation of murine T cell clones can be supported by IL-2 or by IL-4. We present here evidence that glucocorticosteroids differentially affect these two pathways of proliferation. Dexamethasone (DEX) and other corticosteroids were observed to induce autocrine proliferation of the D10.G4.1 Th cell clone (D10) in the presence of the anti-clonotypic antibody 3D3. This effect was inhibited by the anti-murine IL-4 antibody 11B11, indicating that it is mediated by IL-4. Furthermore, on this cell line, representative of the Th2 group of helper cells, DEX had little effect on the proliferation induced by exogenous IL-4 but completely inhibited the growth-promoting effects of IL-2. In contrast, the effects of DEX on the proliferation of the cytotoxic IL-2-dependent CTLL-2 cell line are completely opposite. DEX blocked the IL-4-driven proliferation of CTLL-2 cells, while leaving unaffected their response to IL-2. It is also shown in this study that the effects of glucocorticoids in this system are totally antagonized by the high affinity anti-glucocorticosteroid RU 38486, indicating that they are mediated through the described intracellular glucocorticoid receptor. These data suggest that the growth effects of IL-2 and IL-4 may be mediated by distinct pathways that are strikingly different in their sensitivity to glucocorticoids. In addition, the regulation of lymphokine-dependent proliferation and the response to glucocorticoids appeared very different in helper and cytotoxic cells.     

15d-PGJ2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-kappaB

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural ligand for peroxisome proliferator-activated receptor gamma, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ(2) inhibited Ox-LDL-induced [3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ(2) or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ(2). The suppression of GM-CSF promoter activity by 15d-PGJ(2) and 2-cyclopenten-1-one was mediated through reduction of NF-kappaB binding to GM-CSF promoter. These results suggest that 15d-PGJ(2) inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-kappaB inactivation.     

雌二醇对肺成纤维细胞增殖的调控

本研究观察了雌二醇（Ｅ２）对培养的人胚肺成纤维细胞增殖的直接和间接调控影响。结果显示：Ｅ２可降低肺成纤维细胞的３Ｈ－ＴｄＲ掺入量;Ｅ２与肺泡巨噬细胞（ＡＭ）培养４ｈ的上清液可使肺成纤维细胞的３Ｈ－ＴｄＲ掺入量显著增加;消炎痛预处理不影响Ｅ２的上述作用;Ｅ２与ＡＭ培养１８ｈ可使培养液中内皮素（ＥＴ）的含量显著升高,但培养４ｈ对ＡＭ的ＥＴ分泌的影响不显著。以上结果表明,Ｅ２对肺成纤维细胞的增殖具有直接抑制和通过ＡＭ而间接促进的双向调控作用,其作用与内源性前列腺素无关。Ｅ２间接促进成纤维细胞增殖的机制除与促进ＡＭ的ＥＴ分泌有关外还有其它细胞因子参与。本结果提示,Ｅ２参与肺间质稳态的调控     

Aldosterone stimulates proliferation of cardiac fibroblasts by activating Ki-RasA and MAPK1/2 signaling

Aldosterone plays a pathological role in cardiac fibrosis by directly affecting cardiac fibroblasts. Understanding of the cellular mechanisms of aldosterone action in cardiac fibroblasts, however, is rudimentary. One possibility is that aldosterone promotes proliferation of cardiac fibroblasts by activating specific cellular signaling cascades. The current study tests whether aldosterone stimulates proliferation of isolated adult rat cardiac myofibroblasts (RCF) by activating Kirsten Ras (Ki-RasA) and its effector, the MAPK1/2 cascade. Aldosterone (10 nM) significantly increased RCF proliferation. This action was sensitive to the mineralocorticoid receptor (MR) antagonist spironolactone. Expression of MR in RCF and the whole rat heart was confirmed by immunoblotting. Aldosterone significantly increased absolute and active (GTP bound) Ki-RasA levels in RCF. Aldosterone, in addition, significantly increased phospho-c-Raf and phospho-MAPK1/2. The effects of aldosterone on Ki-RasA and phospho-c-Raf proteins were inhibited by spironolactone but not RU-486, suggesting that aldosterone acts via MR. Inhibitors of MEK1/2 and c-Raf prevented aldosterone-induced activation of MAPK1/2 and proliferation. These results show that aldosterone directly increases RCF proliferation through MR-dependent activation of Ki-RasA and its effector, the MAPK1/2 cascade. Activation of cardiac fibroblasts through such a cascade may play a role in the pathological actions exerted by aldosterone on the heart.     

Novel evidence that testosterone promotes cell proliferation and differentiation via G protein-coupled receptors in the rat L6 skeletal muscle myoblast cell line

Androgens are known to modulate the skeletal muscle proliferation and differentiation processes. Recent in vitro studies have shown that dihydrotestosterone and anabolic steroids have functions in promoting the proliferation and differentiation of the mouse skeletal muscle myoblast C2C12 cell line through the classical androgen receptor (AR) signaling pathway. But there are contradictory reports that androgen plays its roles through the membrane signaling pathways. In the present study, we show that there is no expression of the classical AR in L6 cells both at gene and protein levels. We then investigated the effects of testosterone (T) on L6 cell proliferation and differentiation. The results show that T promotes L6 cell proliferation after a 24 h treatment, which followed by enhancing L6 cell differentiation, but these effects are not inhibited by flutamide (F), an antagonist of intracellular AR. Further, we tested the effect of testosterone covalently bounding to albumin (T-BSA), which does not cross the plasma membrane. The results demonstrate that T-BSA and free T have similar effects on L6 cell proliferation and differentiation, and that these effects involve G protein-coupled receptors and different downstream pathways. The L6 cell proliferation induced by T involves PKC and ERK1/2 signaling pathways and cell differentiation happens via the PKA signaling pathway. These results suggest that T promotes cell proliferation and differentiation via G protein-coupled receptors and different downstream pathways in the L6 cell line, although the related molecular mechanisms need to be elucidated in future studies.     

新基因BCAPP2Ac在膀胱癌中的表达 及其对膀胱癌细胞增殖的影响

为探讨膀胱癌相关蛋白磷酸酶2A催化亚单位（BCAPP2Ac）新基因在膀胱癌组织中的表达及其对膀胱癌细胞增殖的影响,通过合成抗原多肽免疫家兔获得抗BCAPP2Ac多克隆抗体及通过慢病毒感染方式获得稳定表达BCAPP2Ac的膀胱癌细胞.采用实时PCR及免疫组化染色方法检测BCAPP2Ac mRNA和蛋白在膀胱癌组织、癌旁组织及其它多种肿瘤组织中的表达;用细胞增殖实验检测BCAPP2Ac对膀胱癌细胞增殖的影响.实时PCR及免疫组化结果显示,BCAPP2Ac mRNA及蛋白在膀胱癌组织较癌旁组织表达明显下调;过表达BCAPP2Ac能抑制膀胱癌EJ、T24细胞的增殖, 蛋白磷酸酶2A催化结构域缺失（△PP2Ac）能逆转BCAPP2Ac的增殖抑制作用.研究结果提示,新基因BCAPP2Ac能抑制膀胱癌细胞的增殖,PP2Ac结构域在其抑制细胞增殖作用中发挥重要作用.     

Differential signalling pathways for EGF versus PDGF activation of Erk1/2 MAP kinase and cell proliferation in brown pre-adipocytes

Stimulation by both adrenergic and non-adrenergic pathways can induce proliferation of brown pre-adipocytes. To understand the signalling pathways involved in non-adrenergic stimulation of cell proliferation, we examined Erk1/2 activation. In primary cultures of mouse brown pre-adipocytes, both EGF (epidermal growth factor) and PDGF (platelet-derived growth factor) induced Erk1/2 activation. EGF-stimulated Erk1/2 activation involved Src tyrosine kinases, but not PKC or PI3K, whereas in PDGF-induced Erk1/2 activation, PI3K, PKC (probably the atypical ζ isoform) and Src were involved sequentially. Both EGF and PDGF induced PI3K-dependent Akt activation that was not involved in Erk1/2 activation. By comparing effects of signalling inhibitors (wortmannin, SH-6, TPA, Gö6983, PP2, PD98059) on EGF- and PDGF-induced Erk1/2 activation and cell proliferation (³H-thymidine incorporation), we conclude that while the signal transduction pathways initiated by these growth factors are clearly markedly different, their effects on cell proliferation can be fully explained through their stimulation of Erk1/2 activation; thus Erk1/2 is a common, essential step for stimulation of proliferation in these cells.